Tuberculosis (TB) is a major global health problem with an alarming rate of mortality associated with it. It is one of the leading infectious diseases caused by bacteria taking one human life every 15-20 seconds globally. Estimates of 2011 reveal that there are almost 9 million new cases and 1.4 million TB deaths (Global Tuberculosis Report 2012, WHO 2013). The disease is caused by Mycobacterium tuberculosis, a member of the genus Mycobacterium, while in a few cases Mycobacterium bovis has been reported to be the causal organism. More than 100 mycobacterium species are known and among them only a few are pathogenic for humans.
Conventional diagnostic methods include examination of sputum smear under a microscope for acid-fast mycobacteria and radiological reading of lungs. However, in most cases the sputum smear examination turns out to be negative for the bacteria due to early stages of infection and lung changes are not readily visible on an x-rays until several months into the infection.
Diagnosis of mycobacteria-related disease poses difficulties for several reasons which have been recognized by researchers and clinicians over the years. Firstly, these bacteria are in small numbers and are already at a contagious stage when they become detectable by conventional methods. Pulmonary disease caused by different mycobacteria are not readily detectable clinically or radiologically. Detection of organism in culture achieves 100% specificity but the growth of mycobacteria in culture takes about 3-6 weeks (Bates et al, Am. Respir. 134, 415-417, 1986) thus making the process time-consuming. In addition, repeated cultures may be required to ensure success.
Several molecular tests for tuberculosis have been developed in the past. These include the Gen-probe ‘Amplified mycobacterium direct test’ by Abbe et al (J. Clin. Mincrobiol. 31, 3270, 1993), ligase chain reaction (LCR) (J. Clin. Microbiol. 35, 2424, 1997), PCR based Roche Amplicor TB test (J. Clin. Microbiol. 33, 1832, 1995) and IS6110 based detection (J. Clin. Microbiol. 28, 2668, 1990).
U.S. Pat. No. 5,168,039 discloses the IS6110 based detection of M. tuberculosis wherein a repetitive DNA segment specific for members of M. tuberculosis complex is used for the diagnosis. While IS6110 based detection system has been shown to have high level of specificity, there are also reports on false positive detections of 3 to 20% making it unreliable (J. Clin. Microbiol. 32, 277, 1994). In addition, lack of IS6110 sequence in some M. tuberculosis strains may also limit the use of the same for diagnostic tests routinely (Tuber. Lung Dis. 76, 550, 1995). U.S. Pat. No. 7,638,309 provides detection of mycobacteria in clinical specimens in the intergenic region between methyl mycotic acid synthase genes mmaA1 and mmaA2 and the flanking region in mmaA1 and mmaA2 genes.
Thus, it appears that there is a paucity of simple, rapid and reliable tests that can specifically detect M. tuberculosis during early stages of the disease. The present invention has identified ‘signature sequences’ that can differentiate M. tuberculosis from a are number of other mycobacterial DNA. These ‘signature sequences’ are used in detection of early disease in clinical samples of patients.